Substituted pyrido[4′,3′:5,6]pyrazino[1,2-a]indoles as anticancer agents

ABSTRACT

Pyrido[4′,3′:5,6]pyrazino[1,2-a]indole compounds are provided. The compounds exhibit inhibitory activity against CK2 enzyme and may be used as anticancer agents. The compounds have the following general formula I: 
     
       
         
         
             
             
         
       
     
     where R1 represents aryl, heteroaryl, aminoaryl, aminoheteroaryl, aminoalkyl, aminocycloalkyl, aryloxy, heteroaryloxy, or alkoxy; and R2 and R3 represent hydrogen, carboxyl, ester, amide, halogen, alkyl, aryl, hydroxyl, alkoxy, aryloxy, aminoalkyl, aminocycloalkyl, aminoaryl, or aminoheteroaryl.

BACKGROUND 1. Field

The present disclosure relates to the clinical application of novelpyrido[4′,3′:5,6]pyrazino[1,2-a]indole compounds as potentialantitumoral/anti-cancer agents.

2. Description Of The Related Art

The implication of the protein kinase CK2 in various cellular processessuch as cell growth, proliferation, death and differentiation constitutea promising drug target for cancer therapy. The overexpression ofprotein kinase CK2 was reported in multiple cancer types. Recentevidence suggests that CK2 also acts as a suppressor of apoptosis byphosphorylating pro-apoptotic proteins. To date, only one CK2 inhibitorCX-4945 sold under the name of Silmitasertib, an orally active drugdeveloped by Cylene Pharmaceuticals, is currently in clinical use forthe treatment of advanced solid tumors (breast, ovarian, etc.) andmultiple myeloma. The astonishing pharmacological profile of theanti-cancer drug, Silmitasertib, opens the door for the development ofnovel potent and selective CK2 inhibitors for cancer chemotherapy.

Multiple series of CK2 inhibitors have been studied for their activitiesin inhibiting cell growth alone as well as combination with otheranti-proliferation agents (U.S. Pat. Nos. 9,062,043B2, 7,956,064B2;publication WO 2010080170 A1 and WO 2011011199 A1). Combining CK2inhibitors with an anti-cancer agent that prohibits cell growth such asalkylating agents, anti-metabolite, vinca alkaloid, taxane,topoisomerase inhibitors, anti-tumor antibiotics, and tyrosine kinaseinhibitors to treat or ameliorate a neoplastic disorder has beendisclosed in WO 2010080170 A1. Additionally, WO 2010008170 A1 alsodiscloses combining CK2 inhibitors with immunosuppressive macrolide totreat a neoplastic disorder.

Certain CK2 inhibitors are also shown to combine with inhibitors tomolecules that are essential in cell growth pathway such as AKTinhibitors, HDAC inhibitors, HSP90 inhibitors, mTOR inhibitors,PBK/niTGR inhibitors, PDK inhibitors, and antibody targetingtumor/cancer antigen to treat or ameliorating neoplastic disordersand/or inflammatory, autoimmune, or infectious disorders (WO 2011011199A1).

The development of a compound having a novel chemical structuredifferent from that of known CK2 inhibitors and fully satisfiable as adrug for cancer treatment as well as a pharmaceutical compositioncontaining the compound as an active ingredient has been desired. Therequirements of antitumor therapeutics call for constant development ofnew anticancer agents with the aim of generating medicaments that aremore potent and well tolerated. Thus, new antitumoral/anti-cancercompounds solving the aforementioned problems are desired.

SUMMARY

The present subject matter relates the use of novelpyrido[4′,3′:5,6]pyrazino[1,2-a]indole derivatives with improvedpotential for treating various types of cancer and with an increasedprobability of being active against drug resistance cancer cells. Therequirements of antitumor therapeutics call for constant development ofnew anticancer agents with the aim of generating medicaments that aremore potent and well tolerated.

The presently described compounds are not only new but have veryvaluable antitumoral properties. The compounds described hereindemonstrated good to excellent cytotoxic property against a variety ofcancer cell lines. The in vivo antitumoral property of the presentmolecules indicated high potency against colon C38 adenocarcinoma, B16melanoma and leukemia P388 with a T/C greater than 200% with 25-50 mg/kgas an i.p. administration dose. Thepyrido[4′,3′:5,6]pyrazino[1,2-a]indole compounds, showing inhibitoryactivity against CK2 enzyme, are potent anticancer agents and may finduseful applications in cancer chemotherapy for the fight against varioustumors.

The present subject matter provides novelpyrido[4′,3′:5,6]pyrazino[1,2-a]indole compounds, for example,pyrido[4′,3′:5,6]pyrazino[1,2-a]indole compounds of the generalformula/structure I, as useful antitumor agents. The present subjectmatter further provides a process for the synthesis of such compounds,pharmaceutical compositions containing these compounds, and their use intherapy for the treatment of cancer as a sole active agent or incombination with other active ingredients.

In an embodiment, the present subject matter relates to a compoundhaving the formula I:

or a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein: R₁ represents an aryl group, a heteroaryl group, anaminoaryl group, an aminoheteroaryl group, an aminoalkyl group, anaminoalkylaryl group, an aminoalkylheteroaryl group, an aminocycloalkylgroup, an aryloxy group, a heteroaryloxy group, or an alkoxy group,wherein each R₁ group may be optionally substituted with from one tothree substituents independently selected from the group consisting of alinear or branched C₁-C₆ alkyl group, a halogen atom, a linear orbranched C₁-C₆ alkoxy group, a linear or branched C₁-C₆ trihaloalkylgroup, a hydroxy group, an amide group, and a group of formula —NR₄R₅,wherein R₄ and R₅, which may be the same or different, eachindependently represent a hydrogen atom or a linear or branched C₁-C₆alkyl group optionally substituted by a hydroxy group, or R₄ and R₅,together with the nitrogen atom to which they are attached, form anitrogen-containing heterocycle, such nitrogen-containing heterocyclebeing optionally substituted with from one to three substituentsindependently selected from the group consisting of a linear or branchedC₁-C₆ alkyl group, a halogen atom, a linear or branched C₁-C₆ alkoxygroup, a linear or branched C₁-C₆ trihaloalkyl group, and a hydroxygroup; andR₂ and R₃ taken independently can each represent hydrogen, a carboxylgroup, an ester group, an amide group, a halogen atom, a linear orbranched C₁-C₆ alkyl group, an aryl group, a hydroxy group, a linear orbranched C₁-C₆ alkoxy group, an aryloxy group, an aminoalkyl group, anaminocycloalkyl group, an aminoaryl group, and an aminoheteroaryl group,wherein each of R₂ and R₃ taken independently may be optionallysubstituted with from one to three substituents independently selectedfrom the group consisting of a linear or branched C₁-C₆ alkyl group, ahalogen atom, a linear or branched C₁-C₆ alkoxy group, a linear orbranched C₁-C₆ trihaloalkyl group, a hydroxy group, and an amide group,and an amine group.

In another embodiment, the present subject matter relates to a compoundhaving the formula I:

or a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein R₁ is aminophenyl substituted with one or moresubstituents independently selected from the group consisting of astraight or branched C₁-C₆ alkyl, trihalomethyl, halogen, hydroxy,—NR4R5, an amide, and C₁-C₆ alkoxy; an aminoethyl group substituted with—NR₄R₅, wherein R₄ and R₅ are independently a linear C₁-C₆ alkyl group,or R₄ and R₅ are taken together with the nitrogen atom to which they areattached to form an optionally substituted nitrogen-containingheterocycle; an aminomethylphenyl group substituted with a methyl group;or a 2-methylpyridin-4-ylamine group;R₂ is —COOH; andR₃ is hydrogen.

In an embodiment, the present subject matter relates to a compoundselected from the group consisting of:6-((4-chlorophenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (1),6-((4-hydroxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (2),6-((4-fluorophenyl)amino)pyrido[4′,3′:5,6]pyrimido[1,2-a]indole-9-carboxylicacid (3),6-((4-methoxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (4),6-((4-isopropylphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (5),6-((4-(dimethylamino)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (6),6-((4-(trifluoromethyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (7),6-((3-(dimethylcarbamoyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (8),6-(2-methylpyridin-4-yl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (9),6-((3-methylbenzyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (10),6-((2-(dimethylamino)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (11),6-((2-(4-methylpiperazin-1-yl)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (12), and a pharmaceutically acceptable salt, ester, stereoisomer,or solvate thereof.

In an embodiment, the present subject matter relates to a process forthe synthesis of the compounds of formula I, including a number ofspecies or specific structures falling under structural formula I.Further contemplated herein are pharmaceutical compositions containingthese compounds, as well as methods of inhibiting CK2 enzyme activityand of treating various cancers by administering the present compoundsto a patient in need thereof.

These and other features of the present subject matter will becomereadily apparent upon further review of the following specification.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The following definitions are provided for the purpose of understandingthe present subject matter and for construing the appended patentclaims.

Definitions

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including, or comprising specific process steps, itis contemplated that compositions of the present teachings can alsoconsist essentially of, or consist of, the recited components, and thatthe processes of the present teachings can also consist essentially of,or consist of, the recited process steps.

It is noted that, as used in this specification and the appended claims,the singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components, or the element or component can beselected from a group consisting of two or more of the recited elementsor components. Further, it should be understood that elements and/orfeatures of a composition or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes”, “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. In addition, where the use of theterm “about” is before a quantitative value, the present teachings alsoinclude the specific quantitative value itself, unless specificallystated otherwise. As used herein, the term “about” refers to a ±10%variation from the nominal value unless otherwise indicated or inferred.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo, andiodo.

As used herein, “alkyl” refers to a straight-chain or branched saturatedhydrocarbon group. Examples of alkyl groups include methyl (Me), ethyl(Et), propyl (e.g., n-propyl and z′-propyl), butyl (e.g., n-butyl,z′-butyl, sec-butyl, tert-butyl), pentyl groups (e.g., n-pentyl,z′-pentyl, -pentyl), hexyl groups, and the like. In various embodiments,an alkyl group can have 1 to 40 carbon atoms (i.e., C₁-C₄₀ alkyl group),for example, 1-30 carbon atoms (i.e., C₁-C₃₀ alkyl group). In someembodiments, an alkyl group can have 1 to 6 carbon atoms, and can bereferred to as a “lower alkyl group” or a “C₁-C₆ alkyl group”. Examplesof lower alkyl groups include methyl, ethyl, propyl (e.g., n-propyl andz′-propyl), and butyl groups (e.g., n-butyl, z′-butyl, sec-butyl,tert-butyl). In some embodiments, alkyl groups can be substituted asdescribed herein. An alkyl group is generally not substituted withanother alkyl group, an alkenyl group, or an alkynyl group.

As used herein, “alkenyl” refers to a straight-chain or branched alkylgroup having one or more carbon-carbon double bonds. Examples of alkenylgroups include ethenyl, propenyl, butenyl, pentenyl, hexenyl,butadienyl, pentadienyl, hexadienyl groups, and the like. The one ormore carbon-carbon double bonds can be internal (such as in 2-butene) orterminal (such as in 1-butene). In various embodiments, an alkenyl groupcan have 2 to 40 carbon atoms (i.e., C₂—C₄₀ alkenyl group), for example,2 to 20 carbon atoms (i.e., C₂—C₂₀ alkenyl group) or 2 to 6 carbon atoms(i.e., C₂—C₆ alkenyl group). In some embodiments, alkenyl groups can besubstituted as described herein. An alkenyl group is generally notsubstituted with another alkenyl group, an alkyl group, or an alkynylgroup.

The term “substituted alkyl” as used herein refers to an alkyl group inwhich 1 or more (up to about 5, for example about 3) hydrogen atoms isreplaced by a substituent independently selected from the group: —O, —S,acyl, acyloxy, optionally substituted alkoxy, optionally substitutedamino (wherein the amino group may be a cyclic amine), azido, carboxyl,(optionally substituted alkoxy)carbonyl, amido, cyano, optionallysubstituted cycloalkyl, optionally substituted cycloalkenyl, halogen,hydroxyl, nitro, sulfamoyl, sulfanyl, sulfinyl, sulfonyl, and sulfonicacid. Some of the optional substituents for alkyl are hydroxy, halogenexemplified by chloro and bromo, acyl exemplified by methylcarbonyl;alkoxy, and heterocyclyl exemplified by morpholino and piperidino. Otheralkyl substituents as described herein may further be contemplated.

The term “substituted alkenyl” refers to an alkenyl group in which 1 ormore (up to about 5, for example about 3) hydrogen atoms is replaced bya substituent independently selected from those listed above withrespect to a substituted alkyl. Other alkenyl substituents as describedherein may further be contemplated.

As used herein, “heteroatom” refers to an atom of any element other thancarbon or hydrogen and includes, for example, nitrogen, oxygen, silicon,sulfur, phosphorus, and selenium.

As used herein, “aryl” refers to an aromatic monocyclic hydrocarbon ringsystem or a polycyclic ring system in which two or more aromatichydrocarbon rings are fused (i.e., having a bond in common with)together or at least one aromatic monocyclic hydrocarbon ring is fusedto one or more cycloalkyl and/or cycloheteroalkyl rings. An aryl groupcan have 6 to 24 carbon atoms in its ring system (e.g., C₆—C₂₄ arylgroup), which can include multiple fused rings. In some embodiments, apolycyclic aryl group can have 8 to 24 carbon atoms. Any suitable ringposition of the aryl group can be covalently linked to the definedchemical structure. Examples of aryl groups having only aromaticcarbocyclic ring(s) include phenyl, 1-naphthyl (bicyclic), 2-naphthyl(bicyclic), anthracenyl (tricyclic), phenanthrenyl (tricyclic),pentacenyl (pentacyclic), and like groups. Examples of polycyclic ringsystems in which at least one aromatic carbocyclic ring is fused to oneor more cycloalkyl and/or cycloheteroalkyl rings include, among others,benzo derivatives of cyclopentane (i.e., an indanyl group, which is a5,6-bicyclic cycloalkyl/aromatic ring system), cyclohexane (i.e., atetrahydronaphthyl group, which is a 6,6-bicyclic cycloalkyl/aromaticring system), imidazoline (i.e., a benzimidazolinyl group, which is a5,6-bicyclic cycloheteroalkyl/aromatic ring system), and pyran (i.e., achromenyl group, which is a 6,6-bicyclic cycloheteroalkyl/aromatic ringsystem). Other examples of aryl groups include benzodioxanyl,benzodioxolyl, chromanyl, indolinyl groups, and the like. In someembodiments, aryl groups can be substituted as described herein. In someembodiments, an aryl group can have one or more halogen substituents,and can be referred to as a “haloaryl” group. Perhaloaryl groups, i.e.,aryl groups where all of the hydrogen atoms are replaced with halogenatoms (e.g., - C6F5), are included within the definition of “haloaryl”.In certain embodiments, an aryl group is substituted with another arylgroup and can be referred to as a biaryl group. Each of the aryl groupsin the biaryl group can be substituted as disclosed herein.

As used herein, “heteroaryl” refers to an aromatic monocyclic ringsystem containing at least one ring heteroatom selected from oxygen (O),nitrogen (N), sulfur (S), silicon (Si), and selenium (Se) or apolycyclic ring system where at least one of the rings present in thering system is aromatic and contains at least one ring heteroatom.Polycyclic heteroaryl groups include those having two or more heteroarylrings fused together, as well as those having at least one monocyclicheteroaryl ring fused to one or more aromatic carbocyclic rings,non-aromatic carbocyclic rings, and/or non-aromatic cycloheteroalkylrings. A heteroaryl group, as a whole, can have, for example, 5 to 24ring atoms and contain 1-5 ring heteroatoms (i.e., 5-20 memberedheteroaryl group). The heteroaryl group can be attached to the definedchemical structure at any heteroatom or carbon atom that results in astable structure. Generally, heteroaryl rings do not contain O—O, S—S,or S—O bonds. However, one or more N or S atoms in a heteroaryl groupcan be oxidized (e.g., pyridine N-oxide thiophene S-oxide, thiopheneS,S-dioxide). Examples of heteroaryl groups include, for example, the 5-or 6-membered monocyclic and 5-6 bicyclic ring systems shown below:where T is O, S, NH, N-alkyl, N-aryl, N-(arylalkyl) (e.g., N-benzyl),SiH₂, SiH(alkyl), Si(alkyl)₂, SiH(arylalkyl), Si(arylalkyl)₂, orSi(alkyl)(arylalkyl). Examples of such heteroaryl rings includepyrrolyl, furyl, thienyl, pyridyl, pyrimidyl, pyridazinyl, pyrazinyl,triazolyl, tetrazolyl, pyrazolyl, imidazolyl, isothiazolyl, thiazolyl,thiadiazolyl, isoxazolyl, oxazolyl, oxadiazolyl, indolyl, isoindolyl,benzofuryl, benzothienyl, quinolyl, 2-methylquinolyl, isoquinolyl,quinoxalyl, quinazolyl, benzotriazolyl, benzimidazolyl, benzothiazolyl,benzisothiazolyl, benzisoxazolyl, benzoxadiazolyl, benzoxazolyl,cinnolinyl, 1H-indazolyl, 2H-indazolyl, indolizinyl, isobenzofuyl,naphthyridinyl, phthalazinyl, pteridinyl, purinyl, oxazolopyridinyl,thiazolopyridinyl, imidazopyridinyl, furopyridinyl, thienopyridinyl,pyridopyrimidinyl, pyridopyrazinyl, pyridopyridazinyl, thienothiazolyl,thienoxazolyl, thienoimidazolyl groups, and the like. Further examplesof heteroaryl groups include 4,5,6,7-tetrahydroindolyl,tetrahydroquinolinyl, benzothienopyridinyl, benzofuropyridinyl groups,and the like. In some embodiments, heteroaryl groups can be substitutedas described herein.

The term “optional” or “optionally” means that the subsequentlydescribed event or circumstance may or may not occur, and that thedescription includes instances where said event or circumstance occursand instances in which it does not. For example, “optionally substitutedalkyl” means either “alkyl” or “substituted alkyl,” as defined herein.

It will be understood by those skilled in the art with respect to anychemical group containing one or more substituents that such groups arenot intended to introduce any substitution or substitution patterns thatare sterically impractical and/or physically non-feasible.

The term “isomers” or “stereoisomers” as used herein relates tocompounds that have identical molecular formulae but that differ in thearrangement of their atoms in space. Stereoisomers that are not mirrorimages of one another are termed “diastereoisomers” and stereoisomersthat are non-superimposable mirror images are termed “enantiomers,” orsometimes optical isomers. A carbon atom bonded to four non-identicalsubstituents is termed a “chiral center.” Certain compounds herein haveone or more chiral centers and therefore may exist as either individualstereoisomers or as a mixture of stereoisomers. Configurations ofstereoisomers that owe their existence to hindered rotation about doublebonds are differentiated by their prefixes cis and trans (or Z and E),which indicate that the groups are on the same side (cis or Z) or onopposite sides (trans or E) of the double bond in the molecule accordingto the Cahn-Ingold-Prelog rules. All possible stereoisomers arecontemplated herein as individual stereoisomers or as a mixture ofstereoisomers.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which the presently described subject matter pertains.

Where a range of values is provided, for example, concentration ranges,percentage ranges, or ratio ranges, it is understood that eachintervening value, to the tenth of the unit of the lower limit, unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the described subject matter. Theupper and lower limits of these smaller ranges may independently beincluded in the smaller ranges, and such embodiments are alsoencompassed within the described subject matter, subject to anyspecifically excluded limit in the stated range. Where the stated rangeincludes one or both of the limits, ranges excluding either or both ofthose included limits are also included in the described subject matter.

Throughout the application, descriptions of various embodiments use“comprising” language. However, it will be understood by one of skill inthe art, that in some specific instances, an embodiment canalternatively be described using the language “consisting essentiallyof” or “consisting of”.

“Subject” as used herein refers to any animal classified as a mammal,including humans, domestic and farm animals, and zoo, sports, and petcompanion animals such as household pets and other domesticated animalssuch as, but not limited to, cattle, sheep, ferrets, swine, horses,poultry, rabbits, goats, dogs, cats and the like.

“Patient” as used herein refers to a subject in need of treatment of acondition, disorder, or disease, such as an acute or chronic airwaydisorder or disease.

For purposes of better understanding the present teachings and in no waylimiting the scope of the teachings, unless otherwise indicated, allnumbers expressing quantities, percentages or proportions, and othernumerical values used in the specification and claims, are to beunderstood as being modified in all instances by the term “about”.Accordingly, unless indicated to the contrary, the numerical parametersset forth in the following specification and attached claims areapproximations that may vary depending upon the desired propertiessought to be obtained. At the very least, each numerical parametershould at least be construed in light of the number of reportedsignificant digits and by applying ordinary rounding techniques.

In an embodiment, the present subject matter relates to a compoundhaving the formula I:

or a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein: R₁ represents an aryl group, a heteroaryl group, anaminoaryl group, an aminoheteroaryl group, an aminoalkyl group, anaminoalkylaryl group, an aminoalkylheteroaryl group, an aminocycloalkylgroup, an aryloxy group, a heteroaryloxy group, or an alkoxy group,wherein each R₁ group may be optionally substituted with from one tothree substituents independently selected from the group consisting of alinear or branched C₁-C₆ alkyl group, a halogen atom, a linear orbranched C₁-C₆ alkoxy group, a linear or branched C₁-C₆ trihaloalkylgroup, a hydroxy group, an amide group, and a group of formula —NR₄R₅,wherein R₄ and R₅, which may be the same or different, eachindependently represent a hydrogen atom or a linear or branched C₁-C₆alkyl group optionally substituted by a hydroxy group, or R4 and R5,together with the nitrogen atom to which they are attached, form anitrogen-containing heterocycle, such nitrogen-containing heterocyclebeing optionally substituted with from one to three substituentsindependently selected from the group consisting of a linear or branchedC₁-C₆ alkyl group, a halogen atom, a linear or branched C₁-C₆ alkoxygroup, a linear or branched C₁-C₆ trihaloalkyl group, and a hydroxygroup; andR2 and R3 taken independently can each represent hydrogen, a carboxylgroup, an ester group, an amide group, a halogen atom, a linear orbranched C₁-C₆ alkyl group, an aryl group, a hydroxy group, a linear orbranched C₁-C₆ alkoxy group, an aryloxy group, an aminoalkyl group, anaminocycloalkyl group, an aminoaryl group, and an aminoheteroaryl group,wherein each of R₂ and R₃ taken independently may be optionallysubstituted with from one to three substituents independently selectedfrom the group consisting of a linear or branched C₁-C₆ alkyl group, ahalogen atom, a linear or branched C₁-C₆ alkoxy group, a linear orbranched C₁-C₆ trihaloalkyl group, a hydroxy group, and an amide group,and an amine group.

In a further embodiment, the present subject matter relates to compoundsof formula I, wherein R₁ is an optionally substituted aminophenyl group,an optionally substituted —NH(C₁—C₆)alkylphenyl group, an optionallysubstituted —NH(C₁-C₆ alkyl) group, or an optionally substitutedaminoheteroaryl group.

In another embodiment, the present subject matter relates to compoundsof formula I, wherein R₁ is aminophenyl substituted with one or moresubstituents independently selected from the group consisting of astraight or branched C₁-C₆ alkyl, trihalomethyl, halogen, hydroxy,—NR₄R₅, an amide, and C₁-C₆ alkoxy.

In yet another embodiment, the present subject matter relates tocompounds of formula I, wherein R₁ is aminophenyl substituted with oneor more substituents independently selected from the group consisting ofC₁-C₃ alkyl, trifluoromethyl, chlorine, fluorine, hydroxy, dimethylamine, dimethyl amide, and methoxy.

In still yet another embodiment, the present subject matter relates tocompounds of formula I, wherein R₁ is an aminoethyl group substitutedwith —NR₄R₅, wherein R₄ and R₅ are independently a linear C₁-C₆ alkylgroup, or R₄ and R₅ are taken together with the nitrogen atom to whichthey are attached to form an optionally substituted nitrogen-containingheterocycle.

In a further embodiment, the present subject matter relates to compoundsof formula I, wherein R₄ and R₅ are both methyl.

In another embodiment, the present subject matter relates to a compoundof formula I, wherein R₄ and R₅ are taken together with the nitrogenatom to which they are attached to form a 4-methylpiperazin- 1-yl group.

In one embodiment, the present subject matter relates to a compound offormula I, wherein R₁ is an aminomethylphenyl group substituted with amethyl group.

In another embodiment, the present subject matter relates to a compoundof formula I, wherein R₁ is a 2-methylpyridin-4-ylamine group.

In a further embodiment, the present subject matter relates to acompound of formula I, wherein R2 is —COOH and R₃ is hydrogen.

In another embodiment, the present subject matter relates to a compoundhaving the formula I:

or a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof, wherein R₁ is aminophenyl substituted with one or moresubstituents independently selected from the group consisting of astraight or branched C₁-C₆ alkyl, trihalomethyl, halogen, hydroxy,—NR₄R₅, an amide, and C₁-C₆ alkoxy; an aminoethyl group substituted with—NR₄R₅, wherein R₄ and R₅ are independently a linear C₁-C₆ alkyl group,or R₄ and R₅ are taken together with the nitrogen atom to which they areattached to form an optionally substituted nitrogen-containingheterocycle; an aminomethylphenyl group substituted with a methyl group;or a 2-methylpyridin-4-ylamine group;R2 is —COOH; andR3 is hydrogen.

In an embodiment, the present subject matter relates to a compoundselected from the group consisting of:6-((4-chlorophenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (1),6-((4-hydroxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (2),6-((4-fluorophenyl)amino)pyrido[4′,3′:5,6]pyrimido[1,2-a]indole-9-carboxylicacid (3),6-((4-methoxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (4),6-((4-isopropylphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (5),6-((4-(dimethylamino)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (6),6-((4-(trifluoromethyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (7),6-((3-(dimethylcarbamoyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (8),6-(2-methylpyridin-4-yl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (9),6-((3-methylbenzyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (10),6-((2-(dimethylamino)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (11),6-((2-(4-methylpiperazin-1-yl)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (12), and a pharmaceutically acceptable salt, ester, stereoisomer,or solvate thereof.

Said differently, the present subject matter can relate to compounds offormula I selected from the group consisting of:

and a pharmaceutically acceptable salt, ester, stereoisomer, or solvatethereof.

It is to be understood that the present subject matter covers allcombinations of substituent groups referred to herein.

The present compounds may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Accordingly, the present subjectmatter includes all solvates of the present compounds of formula I andpharmaceutically acceptable stereoisomers, esters, and/or salts thereof.Hydrates are one example of such solvates.

Further, the present subject matter includes all mixtures of possiblestereoisomers of the embodied compounds, independent of the ratio,including the racemates.

Salts of the present compounds, or the salts of the stereoisomersthereof, include all inorganic and organic acid addition salts and saltswith bases, especially all pharmaceutically acceptable inorganic andorganic acid addition salts and salts with bases, particularly allpharmaceutically acceptable inorganic and organic acid addition saltsand salts with bases customarily used in pharmacy.

Examples of acid addition salts include, but are not limited to,hydrochlorides, hydrobromides, phosphates, nitrates, sulfates, acetates,trifluoroacetates, citrates, D-gluconates, benzoates,2-(4-hydroxy-benzoyl)benzoates, butyrates, subsalicylates, maleates,laurates, malates, lactates, fumarates, succinates, oxalates, tartrates,stearates, benzenesulfonates (besilates), toluenesulfonates (tosilates),methanesulfonates (mesilates) and 3-hydroxy-2-naphthoates.

Examples of salts with bases include, but are not limited to, lithium,sodium, potassium, calcium, aluminum, magnesium, titanium, ammonium,meglumine and guanidinium salts. The salts include water-insoluble and,particularly, water-soluble salts.

The present compounds, the salts, the stereoisomers and the salts of thestereoisomers thereof may contain, e.g., when isolated in crystallineform, varying amounts of solvents. Included within the present scopeare, therefore, all solvates of the compounds of formula I, as well asthe solvates of the salts, the stereoisomers and the salts of thestereoisomers of the compounds of formula I.

The present compounds may be isolated and purified in a manner known perse, e.g., by distilling off the solvent in vacuo and recrystallizing theresidue obtained from a suitable solvent or subjecting it to one of thecustomary purification methods, such as column chromatography on asuitable support material.

Salts of the compounds of formula I and the stereoisomers thereof can beobtained by dissolving the free compound in a suitable solvent (by wayof non-limiting example, a ketone such as acetone, methylethylketone ormethylisobutylketone; an ether such as diethyl ether, tetrahydrofuraneor dioxane; a chlorinated hydrocarbon such as methylene chloride orchloroform; a low molecular weight aliphatic alcohol such as methanol,ethanol or isopropanol; a low molecular weight aliphatic ester such asethyl acetate or isopropyl acetate; or water) which contains the desiredacid or base, or to which the desired acid or base is then added. Theacid or base can be employed in salt preparation, depending on whether amono- or polybasic acid or base is concerned and depending on which saltis desired, in an equimolar quantitative ratio or one differingtherefrom. The salts are obtained by filtering, reprecipitating,precipitating with a non-solvent for the salt or by evaporating thesolvent. Salts obtained can be converted into the free compounds which,in turn, can be converted into salts. In this manner, pharmaceuticallyunacceptable salts, which can be obtained, for example, as processproducts in the manufacturing on an industrial scale, can be convertedinto pharmaceutically acceptable salts by processes known to the personskilled in the art.

Pure diastereomers and pure enantiomers of the present compounds can beobtained, e.g., by asymmetric synthesis, by using chiral startingcompounds in synthesis and by splitting up enantiomeric anddiastereomeric mixtures obtained in synthesis. Preferably, the purediastereomeric and pure enantiomeric compounds are obtained by usingchiral starting compounds in synthesis.

Enantiomeric and diastereomeric mixtures can be split up into the pureenantiomers and pure diastereomers by methods known to a person skilledin the art. Preferably, diastereomeric mixtures are separated bycrystallization, in particular fractional crystallization, orchromatography. Enantiomeric mixtures can be separated, e.g., by formingdiastereomers with a chiral auxiliary agent, resolving the diastereomersobtained and removing the chiral auxiliary agent. As chiral auxiliaryagents, for example, chiral acids can be used to separate enantiomericbases and chiral bases can be used to separate enantiomeric acids viaformation of diastereomeric salts. Furthermore, diastereomericderivatives such as diastereomeric esters can be formed fromenantiomeric mixtures of alcohols or enantiomeric mixtures of acids,respectively, using chiral acids or chiral alcohols, respectively, aschiral auxiliary agents. Additionally, diastereomeric complexes ordiastereomeric clathrates may be used for separating enantiomericmixtures. Alternatively, enantiomeric mixtures can be split up usingchiral separating columns in chromatography. Another suitable method forthe isolation of enantiomers is enzymatic separation.

In one embodiment, the present compounds can be prepared according tothe following general synthetic pathway. Specifically, synthesiscommences with heating a mixture of dimethyl 1H-indole-2,5-dicarboxylateI, 3-iodo-4-nitropyridine II, CuI, metformin hydrochloride, Cs₂CO₃, andDMF. After cooling to room temperature, dilution, filtering, andconcentration, the resulting residue is purified to afford dimethyl1-(4-nitropyridin-3-yl)-1H-indole-2,5-dicarboxylate III as shown inScheme 1.

Next a mixture of dimethyl1-(4-nitropyridin-3-yl)-1H-indole-2,5-dicarboxylate III and Pd on carbonis stirred. After removing the catalyst by filtration, the filtrate isevaporated. The resulting crude mixture is purified to provide methyl6-oxo-5,6-dihydropyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylateIV, which is then chlorinated in CH₂Cl₂with SOCl₂.The organic layer iswashed, dried, filtrated, evaporated, and crystallized to provide thecorresponding 6-chloro product IV as outlined in Scheme 2.

The final step of the synthesis involves mixing the 6-chloro derivativeand a variety of amines. The solid obtained is filtrated and washed. Amixture of methyl 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylate derivativesand KOH is then heated, cooled, filtrated, washed, and recrystallized toobtain the corresponding 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V(examples 1-12) as shown in Scheme 3.

In another embodiment, the present subject matter is directed topharmaceutical compositions comprising a therapeutically effectiveamount of the compounds as described herein together with one or morepharmaceutically acceptable carriers, excipients, or vehicles. In someembodiments, the present compositions can be used for combinationtherapy, where other therapeutic and/or prophylactic ingredients can beincluded therein.

The present subject matter further relates to a pharmaceuticalcomposition, which comprises at least one of the present compoundstogether with at least one pharmaceutically acceptable auxiliary.

In an embodiment, the pharmaceutical composition comprises one or two ofthe present compounds, or one of the present compounds.

Non-limiting examples of suitable excipients, carriers, or vehiclesuseful herein include liquids such as water, saline, glycerol,polyethyleneglycol, hyaluronic acid, ethanol, and the like. Suitableexcipients for nonliquid formulations are also known to those of skillin the art. A thorough discussion of pharmaceutically acceptableexcipients and salts useful herein is available in Remington'sPharmaceutical Sciences, 18th Edition. Easton, Pa., Mack PublishingCompany, 1990, the entire contents of which are incorporated byreference herein.

The present compounds are typically administered at a therapeutically orpharmaceutically effective dosage, e.g., a dosage sufficient to providetreatment for cancer. Administration of the compounds or pharmaceuticalcompositions thereof can be by any method that delivers the compoundssystemically and/or locally. These methods include oral routes,parenteral routes, intraduodenal routes, and the like.

While human dosage levels have yet to be optimized for the presentcompounds, generally, a daily dose is from about 0.01 to 10.0 mg/kg ofbody weight, for example about 0.1 to 5.0 mg/kg of body weight. Theprecise effective amount will vary from subject to subject and willdepend upon the species, age, the subject's size and health, the natureand extent of the condition being treated, recommendations of thetreating physician, and the therapeutics or combination of therapeuticsselected for administration. The subject may be administered as manydoses as is required to reduce and/or alleviate the signs, symptoms, orcauses of the disease or disorder in question, or bring about any otherdesired alteration of a biological system.

In employing the present compounds for treatment of cancer, anypharmaceutically acceptable mode of administration can be used withother pharmaceutically acceptable excipients, including solid,semi-solid, liquid or aerosol dosage forms, such as, for example,tablets, capsules, powders, liquids, suspensions, suppositories,aerosols or the like. The present compounds can also be administered insustained or controlled release dosage forms, including depotinjections, osmotic pumps, pills, transdermal (includingelectrotransport) patches, and the like, for the prolongedadministration of the compound at a predetermined rate, preferably inunit dosage forms suitable for single administration of precise dosages.

The present compounds may also be administered as compositions preparedas foods for foods or animals, including medical foods, functional food,special nutrition foods and dietary supplements. A “medical food” is aproduct prescribed by a physician that is intended for the specificdietary management of a disorder or health condition for whichdistinctive nutritional requirements exist and may include formulationsfed through a feeding tube (referred to as enteral administration orgavage administration).

A “dietary supplement” shall mean a product that is intended tosupplement the human diet and may be provided in the form of a pill,capsule, tablet, or like formulation. By way of non-limiting example, adietary supplement may include one or more of the following dietaryingredients: vitamins, minerals, herbs, botanicals, amino acids, anddietary substances intended to supplement the diet by increasing totaldietary intake, or a concentrate, metabolite, constituent, extract, orcombinations of these ingredients, not intended as a conventional foodor as the sole item of a meal or diet. Dietary supplements may also beincorporated into foodstuffs, such as functional foods designed topromote control of glucose levels. A “functional food” is an ordinaryfood that has one or more components or ingredients incorporated into itto give a specific medical or physiological benefit, other than a purelynutritional effect. “Special nutrition food”means ingredients designedfor a particular diet related to conditions or to support treatment ofnutritional deficiencies.

Generally, depending on the intended mode of administration, thepharmaceutically acceptable composition will contain about 0.1% to 90%,for example about 0.5% to 50%, by weight of a compound or salt of thepresent compounds, the remainder being suitable pharmaceuticalexcipients, carriers, etc.

One manner of administration for the conditions detailed above is oral,using a convenient daily dosage regimen which can be adjusted accordingto the degree of affliction. For such oral administration, apharmaceutically acceptable, non-toxic composition is formed by theincorporation of any of the normally employed excipients, such as, forexample, mannitol, lactose, starch, magnesium stearate, sodiumsaccharine, talcum, cellulose, sodium crosscarmellose, glucose, gelatin,sucrose, magnesium carbonate, and the like. Such compositions take theform of solutions, suspensions, tablets, dispersible tablets, pills,capsules, powders, sustained release formulations and the like.

The present compositions may take the form of a pill or tablet and thusthe composition may contain, along with the active ingredient, a diluentsuch as lactose, sucrose, dicalcium phosphate, or the like; a lubricantsuch as magnesium stearate or the like; and a binder such as starch, gumacacia, polyvinylpyrrolidine, gelatin, cellulose and derivativesthereof, and the like.

Liquid pharmaceutically administrable compositions can, for example, beprepared by dissolving, dispersing, etc. an active compound as definedabove and optional pharmaceutical adjuvants in a carrier, such as, forexample, water, saline, aqueous dextrose, glycerol, glycols, ethanol,and the like, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, sodium acetate, sodium citrate, cyclodextrinederivatives, sorbitan monolaurate, triethanolamine acetate,triethanolamine oleate, etc.

For oral administration, a pharmaceutically acceptable non-toxiccomposition may be formed by the incorporation of any normally employedexcipients, such as, for example, pharmaceutical grades of mannitol,lactose, starch, magnesium stearate, talcum, cellulose derivatives,sodium crosscarmellose, glucose, sucrose, magnesium carbonate, sodiumsaccharin, talcum and the like. Such compositions take the form ofsolutions, suspensions, tablets, capsules, powders, sustained releaseformulations and the like.

For a solid dosage form, a solution or suspension in, for example,propylene carbonate, vegetable oils or triglycerides, may beencapsulated in a gelatin capsule. Such diester solutions, and thepreparation and encapsulation thereof, are disclosed in U.S. Pat. Nos.4,328,245; 4,409,239; and 4,410,545, the contents of each of which areincorporated herein by reference. For a liquid dosage form, thesolution, e.g., in a polyethylene glycol, may be diluted with asufficient quantity of a pharmaceutically acceptable liquid carrier,e.g., water, to be easily measured for administration.

Alternatively, liquid or semi-solid oral formulations may be prepared bydissolving or dispersing the active compound or salt in vegetable oils,glycols, triglycerides, propylene glycol esters (e.g., propylenecarbonate) and the like, and encapsulating these solutions orsuspensions in hard or soft gelatin capsule shells.

Other useful formulations include those set forth in U.S. Pat. Nos. Re.28,819 and 4,358,603, the contents of each of which are herebyincorporated by reference.

Another manner of administration is parenteral administration, generallycharacterized by injection, either subcutaneously, intramuscularly orintravenously. Injectables can be prepared in conventional forms, eitheras liquid solutions or suspensions, solid forms suitable for solution orsuspension in liquid prior to injection, or as emulsions. Suitableexcipients are, for example, water, saline, dextrose, glycerol, ethanolor the like. In addition, if desired, the pharmaceutical compositions tobe administered may also contain minor amounts of non-toxic auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,solubility enhancers, and the like, such as for example, sodium acetate,sorbitan monolaurate, triethanolamine oleate, cyclodextrins, etc.

Another approach for parenteral administration employs the implantationof a slow- release or sustained-release system, such that a constantlevel of dosage is maintained. The percentage of active compoundcontained in such parenteral compositions is highly dependent on thespecific nature thereof, as well as the activity of the compound and theneeds of the subject. However, percentages of active ingredient of 0.01%to 10% in solution are employable and will be higher if the compositionis a solid which will be subsequently diluted to the above percentages.The composition may comprise 0.2% to 2% of the active agent in solution.

Nasal solutions of the active compound alone or in combination withother pharmaceutically acceptable excipients can also be administered.

Formulations of the active compound or a salt may also be administeredto the respiratory tract as an aerosol or solution for a nebulizer, oras a microfine powder for insufflation, alone or in combination with aninert carrier such as lactose. In such a case, the particles of theformulation have diameters of less than 50 microns, for example lessthan 10 microns.

The present compounds have valuable pharmaceutical properties, whichmake them commercially utilizable. Accordingly, the present subjectmatter further relates to use of the present compounds for the treatmentof diseases such as cancers. Similarly, the present compounds can beused to inhibit CK2 enzyme activity in a patient.

In another embodiment of the present subject matter, the aforementionedcompound derivatives demonstrated in vitro anticancer action againsthuman cancer cell lines such as MCF7 and MDA-MB-231 (breast cancer),H1299 (lung cancer), PC3 (prostate cancer), HCT116 (colon cancer), A375(melanoma), MIAPaCa2 (pancreatic cancer), and HL60 (leukemia).Accordingly, the present subject matter relates to methods of treating acancer in a patient by administering one or more of the compoundspresented herein to a patient in need thereof. In certain embodiments,the cancer treatable with the present compounds is one or more selectedfrom the group consisting of leukemia, melanoma, colon cancer, prostatecancer, lung cancer, pancreatic cancer, and breast cancer.

Accordingly, in an embodiment of the present subject matter, thepyrido[4′,3′:5,6]pyrazino[1,2-a]indole derivatives, or the 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acidderivatives, as described herein engaged for in vitro study towardshuman cancer cell lines can display an IC₅₀ with a nano to micromolarconcentration range when exposed to a period of at least 96 hrs. Forexample, a present compound engaged for in vitro study against PC3(prostate) cancer cell lines can display an IC₅₀ concentration of 1.6 μMat an exposure period of at least 96 hrs.

In another embodiment, a present compound engaged for in vitro studyagainst HCT116 (colon) cancer cell lines can display an IC₅₀concentration of 1.9 μM at an exposure period of at least 96 hrs.

In a further embodiment, a present compound engaged for in vitro studyagainst A375 (melanoma) cancer cell lines can display an IC₅₀concentration of 2.5 μM at an exposure period of at least 96 hrs.

In an embodiment, a present compound engaged for in vitro study againstH1299 (lung) cancer cell lines can display an IC₅₀ concentration of 3.1μM at an exposure period of at least 96 hrs.

In another embodiment, a present compound engaged for in vitro studyagainst MIAPaCa-2 (pancreas) cancer cell lines can display an IC₅₀concentration of 0.9 μM at an exposure period of at least 96 hrs.

In a further embodiment, a present compound engaged for in vitro studyagainst HL60 (leukemia) cancer cell lines can display an IC₅₀concentration of 2.8 μM at an exposure period of at least 96 hrs.

In one embodiment, a present compound engaged for in vitro study againstMCF7 (breast) cancer cell lines can display an IC₅₀ concentration of 7.8μM at an exposure period of at least 96 hrs.

In another embodiment, a present compound engaged for in vitro studyagainst MDA-MB-231 (breast) cancer cell lines can display an IC₅₀concentration of 6.1 μM at an exposure period of at least 96 hrs.

The present subject matter further relates to a method of treating orpreventing a disease comprising administering to a patient in needthereof a therapeutically effective amount of at least one of thecompounds herein.

In particular, the present subject matter relates to a method oftreating one of the above-mentioned diseases or disorders comprisingadministering to a patient in need thereof a therapeutically effectiveamount of at least one of the compounds herein.

In the above methods, the patient is preferably a mammal, morepreferably a human. Furthermore, in the above methods, at least one ofthe present compounds can be used. In an embodiment, one or two of thepresent compounds are used, or one of the present compounds is used.Similarly, one or more of the present compounds can be used incombination therapy with one or more additional active agents.

The following examples relate to various methods of manufacturingcertain specific compounds as described herein. All compound numbersexpressed herein are with reference to the synthetic pathway figuresshown above.

EXAMPLES Example 1 Preparation of dimethyl1-(4-nitropyridin-3-yl)-1H-indole-2,5-dicarboxylate III

A mixture of dimethyl 1H-indole-2,5-dicarboxylate I (5 mmol),3-iodo-4-nitropyridine II (5 mmol), CuI (0.5 mmol), metforminhydrochloride (1 mmol), Cs₂CO₃ (10 mmol), and DMF (10 mL) was heated to130° C. for 8 hours under N₂ atmosphere. After cooling to roomtemperature, the reaction mixture was diluted with EtOAc (20 mL). Thesolid was removed by filter, and the filtrate was washed with water andbrine. The organic layer was dried over MgSO₄, filtered, andconcentrated under reduced pressure. The resulting residue was purifiedby column chromatography to afford dimethyl1-(4-nitropyridin-3-yl)-1H-indole-2,5-dicarboxylate III.

Elemental Analysis: Calculated C, 57.47; H, 3.69; N, 11.83; Found C,57.45; H, 3.73; N, 11.80

Example 2 Preparation of methyl6-chloropyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylate IV

A mixture of dimethyl1-(4-nitropyridin-3-yl)-1H-indole-2,5-dicarboxylate III (3 mmol) and 5%Pd on carbon (15 mg) in formic acid 5 mL was stirred at room temperatureunder nitrogen for 16 hours. After removing the catalyst by filtration,the filtrate was evaporated under reduced pressure. The resulting crudemixture was purified by silica gel chromatography to provide theexpected product methyl6-oxo-5,6-dihydropyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylate IVwhich was engaged in the chlorination step.

To a mixture of methyl6-oxo-5,6-dihydropyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylate (2mmol) in CH₂Cl₂ (10 mL) were added SOCl₂ (3 mmol) and a drop of DMF. Thereaction mixture was stirred at room temperature for 4 hours. Theorganic layer was washed with ammonium chloride solution. The organiclayer was dried over MgSO₄, filtrated, evaporated and crystallized toprovide the corresponding 6-chloro product IV.

Elemental Analysis: Calculated C, 61.65; H, 3.23; N, 13.48; Found C,61.62; H, 3.68; N, 13.51

Example 3 General Procedure for the Preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V

A mixture of 6-chloro derivative (1 mmol) and a variety of amines (2mmol) in 5 mL 2-ethoxyethanol was heated at reflux for 6 hours. Aftercooling to room temperature, the solid obtained was filtrated, washedwith diethyl ether, and was engaged in the next step without furtherpurification.

A mixture of methyl 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylate derivatives (1mmol) and KOH (3 mmol) in 5 mL THF was heated at reflux for 2 hours.After cooling to room temperature, a solution of 10% HCl was added andthe resulting precipitate was filtrated, washed with water, andrecrystallized to furnish the corresponding 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V(examples 1-12).

Example 4 Preparation of6-((4-chlorophenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (1)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-chloroaniline as the amine.

Elemental Analysis: Calculated C, 64.87; H, 3.37; N, 14.41; Found C,64.91; H, 3.40; N, 14.38.

Example 5 Preparation of6-((4-hydroxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (2)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-hydroxyaniline as the amine.

Elemental Analysis: Calculated C, 66.00; H, 3.50; N, 20.25; Found C,65.95; H, 3.52; N, 20.21.

Example 6 Preparation of6-((4-fluorophenyl)amino)pyrido[4′,3′:5,6]pyrimido[1,2-a]indole-9-carboxylicacid (3)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-fluoroaniline as the amine.

Elemental Analysis: Calculated C, 67.74; H, 3.52; F, 5.10; N, 15.05;Found C, 67.69; H, 3.58; N, 15.00.

Example 7 Preparation of6-((4-methoxyphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (4)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-methoxylaniline as the amine.

Elemental Analysis: Calculated C, 68.74; H, 4.20; N, 14.58; Found C,68.69; H, 4.17; N, 14.63.

Example 8 Preparation of6-((4-isopropylphenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (5)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-isopropylaniline as the amine.

Elemental Analysis: Calculated C, 72.71; H, 5.09; N, 14.13; Found C,72.74; H, 5.12; N, 14.09.

Example 9 Preparation of6-((4-(dimethylamino)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (6)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-dimethylaminoaniline as the amine.

Elemental Analysis: Calculated C, 69.51; H, 4.82; N, 17.62; Found C,69.46; H, 4.79; N, 17.68.

Example 10 Preparation of6-((4-(trifluoromethyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (7)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 4-trifluoroaniline as the amine.

Elemental Analysis: Calculated C, 62.56; H, 3.10; N, 13.27; Found C,62.51; H, 3.08; N, 13.23.

Example 11 Preparation of6-((3-(dimethylcarbamoyl)phenyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (8)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 3-amino-N,N-dimethylbenzamide as the amine.

Elemental Analysis: Calculated C, 67.76; H, 4.50; N, 16.46; Found C,67.74; H, 4.52; N, 16.50.

Example 12 Preparation of6-((2-methylpyridin-4-yl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (9)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 2-methylpyridin-4-amine as the amine.

Elemental Analysis: Calculated C, 68.28; H, 4.09; N, 18.96; Found C,59.33; H, 4.14; N, 19.02.

Example 13

Preparation of6-((3-methylbenzyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (10)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 3-methylbenzylamine as the amine.

Elemental Analysis: Calculated C, 72.24; H, 4.74; N, 14.65; Found C,72.23; H, 4.78; N, 14.63.

Example 14 Preparation of6-((2-(dimethylamino)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (11)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using N1,N1-dimethylethane-1,2-diamine as the amine.

Elemental Analysis: Calculated C, 65.32; H, 5.48; N, 20.04; Found C,65.26; H, 5.52; N, 20.09.

Example 15 Preparation of6-((2-(4-methylpiperazin-1-yl)ethyl)amino)pyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylicacid (12)

The expected product was obtained in accordance with the generalprocedure for the preparation of 6-substitutedaminopyrido[4′,3′:5,6]pyrazino[1,2-a]indole-9-carboxylic acid V ofExample 3 using 3-(4-methylpiperazin-1-yl)ethan-1-amine as the amine.

Elemental Analysis: Calculated C, 65.33; H, 5.98; N, 20.78; Found C,65.29; H, 6.02; N, 20.83.

Pharmacological Activity Example 16 In vitro Cytotoxic Activity Assay

Compounds 1-12 were screened for their in vitro cytotoxic activityutilizing a 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay against selected cancer human cell lines consistingof PC3 (prostate), HCT116 (colon), A375 (melanoma), H1299 (lung),MIAPaCa-2 (pancreas), HL60 (leukemia), MCF7 (breast), MDA-MB-231(breast) (T. Mosmann, J. Immunol. Meth., 1983, 65, 55-63). The cellswere cultured at 37° C. in RMPI1640 medium supplemented with 10% fetalbovine serum, 50 IU/mL penicillin, and 50 μg/mL streptomycin in a 5% CO₂incubator. All cells were sub-cultured 3 times/week by trypsinisation.Viable cells were seeded and allowed to adhere for 12 h before a testdrug was added in 96-well plates at an initial density of 1.0×105cells/mL. Tumour cell lines were separately exposed to variousconcentrations of the tested compounds followed by incubation at atemperature of 37° C. during 96 h inside a medium of fresh RMPI 1640.Cells were subsequently incubated at 37° C. using MTT at 0.5 mg/mLduring 4 h. After removal of supernatant, formazan crystals weredissolved in isopropanol and the optical density was measured at 570 nm.CX-4945 was used as a positive control.

By way of example, the compound (1) displayed promisinganti-proliferative activity against human cancer cells as reported inTable 1.

TABLE 1 Cytotoxicity activity of compound of Compound (1) and CX-4945 onvarious human cancer cells ^(a) Cancer cell lines Compound (1) CX-4945PC3 (prostate) 1.6 2.0 HCT116 (colon) 1.9 2.3 A375 (melanoma) 2.5 4.1H1299 (lung) 3.1 2.3 MIAPaCa-2 (pancreas) 0.9 1.0 HL60 (leukemia) 2.83.7 MCF7 (breast) 7.8 9.1 MDA-MB-231 (breast) 6.1 6.2 ^(a) Cells wereexposed for 96 hours and the number of viable cells was measured usingthe MTS reagent. IC₅₀ values were calculated as the concentration ofcompound eliciting a 50% inhibition of cell proliferation expressed inμM.

The biological results demonstrated that the compound (1) displayedpromising in vitro anti-proliferative activity against various humancancer cell lines similar to that of CX-4945 used as reference drug.

Example 17 Evaluation of Inhibitory Activity on Protein Kinase CK2

CK2 Kinase Assay was conducted using the protocol as described in PierreF. et al; J. Med. Chem. 2011, 54, 635-654. The tested compounds inaqueous solution were added at a volume of 10 μL to a reaction mixturecomprising 10 μL of assay dilution buffer (ADB; 20 mM MOPS, pH 7.2, 25mM β-glycerol phosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mMdithiothreitol), 10 μL of substrate peptide (RRRDDDSDDD, dissolved inADB at a concentration of 1 mM), 10 μL of recombinant human CK2(RRfββ-holoenzyme, 25 ng dissolved in ADB; Millipore). Reactions wereinitiated by the addition of 10 μL of ATP solution (90% 75 mMMgCl₂, 75μM ATP (final ATP concentration:15 μM) dissolved in ADB; 10% [γ-33P] ATP(stock 1mCi/100 μL; 3000 Ci/mmol (Perkin-Elmer) and maintained for 10min at 30° C. The reactions were quenched with 100 μL of 0.75%phosphoric acid and then transferred to and filtered through a phosphorcellulose filter plate (Millipore). After washing each well five timeswith 0.75% phosphoric acid, the plate was dried under vacuum for 5 minand, following the addition of 15 μL of scintillation fluid to eachwell, the residual radioactivity was measured using a Wallacluminescence counter. The IC₅₀ values were derived from eightconcentrations of test inhibitors.

The biological results demonstrated that the present compounds possessedfavourable CK2 inhibition with IC₅₀ at a nanomolar concentration range.

By way of example, the compound (1) displayed promising protein kinaseCK2 activity with an IC₅₀ of 12 nM, while in the same experimentalcondition, the reference control CX-4945 inhibited protein kinase CK2activity of 1 nM.

It is to be understood that the pyrido[4′,3′:5,6]pyrazino[1,2-a]indolederivatives are not limited to the specific embodiments described above,but encompasses any and all embodiments within the scope of the genericlanguage of the following claims enabled by the embodiments describedherein, or otherwise shown in the drawings or described above in termssufficient to enable one of ordinary skill in the art to make and usethe claimed subject matter.

We claim:
 1. A compound having the formula I:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:R₁ is NH(alkyl), NH(alkylenearyl), NH(alkyleneheteroaryl),NH(cycloalkyl), NH(aryl), NH(heteroaryl), O(alkyl), O(aryl),O(heteroaryl), aryl, or heteroaryl, wherein any alkyl, aryl ofalkylenearyl, heteroaryl of alkyleneheteroaryl, cycloalkyl, aryl, orheteroaryl is optionally substituted with one, two, or threesubstituents independently selected from the group consisting ofhalogen, C₁-C₆ alkyl, C₁-C₆ trihaloalkyl, C(O)NR₄R₅, NR₄R₅, OH, andOC₁-C₆ alkyl; R₂ is H, halogen, C₁-C₆ alkyl, C(O)NR₄R₅, C(O)OH,C(O)O(alkyl), C(O)O(cycloalkyl), C(O)O(heterocyclyl), C(O)O(aryl),C(O)O(heteroaryl), NH(alkyl), NH(cycloalkyl), NH(aryl), NH(heteroaryl),OH, OC₁-C₆ alkyl, O(aryl), or aryl, wherein any alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl is optionally substituted with one,two, or three substituents independently selected from the groupconsisting of halogen, C₁-C₆ alkyl, C₁-C₆ trihaloalkyl, C(O)NR₄R₅, NH₂,OH, and OC₁-C₆ alkyl; R₃ is H, halogen, C₁-C₆ alkyl, C(O)NR₄R₅, C(O)OH,C(O)O(alkyl), C(O)O(cycloalkyl), C(O)O(heterocyclyl), C(O)O(aryl),C(O)O(heteroaryl), NH(alkyl), NH(cycloalkyl), NH(aryl), NH(heteroaryl),OH, OC₁-C₆ alkyl, O(aryl), or aryl, wherein any alkyl, cycloalkyl,heterocyclyl, aryl, or heteroaryl is optionally substituted with one,two, or three substituents independently selected from the groupconsisting of halogen, C₁-C₆ alkyl, C₁-C₆ trihaloalkyl, C(O)NR₄R₅, NH₂,OH, and OC₁-C₆ alkyl; each R₄ is independently H or C₁-C₆ alkyl, whereineach C₁-C₆ alkyl is optionally and independently substituted with one ormore hydroxy substituents; and each R₅ is independently H or C₁-C₆alkyl, wherein each C₁-C₆ alkyl is optionally and independentlysubstituted with one or more hydroxy substituents; or any R₄ and R₅,taken together with the nitrogen atom to which they are attached,independently forms a nitrogen-containing heterocyclyl, wherein eachnitrogen-containing heterocyclyl is optionally and independentlysubstituted with one, two, or three substituents independently selectedfrom the group consisting of halogen, C₁-C₆ alkyl, C₁-C₆ trihaloalkyl,OH, and OC₁-C₆ alkyl.
 2. The compound of claim 1, or a pharmaceuticallyacceptable salt or stereoisomer thereof, wherein R₁ is NH(C₁-C₆ alkyl),NH(C₁-C₆ alkylenephenyl), NH(phenyl), or NH(heteroaryl), wherein theC₁-C₆ alkyl, phenyl of C₁-C₆ alkylenephenyl, phenyl, or heteroaryl isoptionally substituted with one, two, or three substituentsindependently selected from the group consisting of halogen, C₁-C₆alkyl, C₁-C₆ trihaloalkyl, C(O)NR₄R₅, NR₄R₅, OH, and OC₁-C₆ alkyl. 3.The compound of claim 2, or a pharmaceutically acceptable salt orstereoisomer thereof, wherein: R₁ is NHCH₂CH₃, wherein the CH₂CH₃ issubstituted with one NR₄R₅ substituent; R₄ is C₁-C₆ alkyl; and R₅ isC₁-C₆ alkyl; or R₄ and R₅, taken together with the nitrogen atom towhich they are attached, form a nitrogen-containing heterocyclyl,wherein the nitrogen-containing heterocyclyl is optionally substitutedwith one, two, or three substituents independently selected from thegroup consisting of halogen, C₁-C₆ alkyl, C₁-C₆ trihaloalkyl, OH, andOC₁-C₆ alkyl.
 4. The compound of claim 3, or a pharmaceuticallyacceptable salt or stereoisomer thereof, wherein: R₄ is CH₃; and R₅ isCH₃; or R₄ and R₅, taken together with the nitrogen atom to which theyare attached, form 4-methylpiperazin-1-yl.
 5. The compound of claim 2,or a pharmaceutically acceptable salt or stereoisomer thereof, whereinR₁ is NHCH₂phenyl, wherein the phenyl of CH₂phenyl is substituted withone CH₃ substituent.
 6. The compound of claim 2, or a pharmaceuticallyacceptable salt or stereoisomer thereof, wherein R₁ is NH(phenyl),wherein the phenyl is optionally substituted with one, two, or threesubstituents independently selected from the group consisting ofhalogen, C₁-C₆ alkyl, C(halo)₃, C(O)NR₄R₅, NR₄R₅, OH, and OC₁-C₆ alkyl.7. The compound of claim 6, or a pharmaceutically acceptable salt orstereoisomer thereof, wherein R₁ is NH(phenyl), wherein the phenyl isoptionally substituted with one, two, or three substituentsindependently selected from the group consisting of F, Cl, C₁-C₃ alkyl,CF₃, C(O)N(CH₃)₂, N(CH₃)₂, OH, and OCH₃.
 8. The compound of claim 2, ora pharmaceutically acceptable salt or stereoisomer thereof, wherein R₁is NH(2-methylpyridin-4-yl).
 9. The compound of claim 1, or apharmaceutically acceptable salt or stereoisomer thereof, wherein: R₂ isC(O)OH; and R₅ is H.
 10. The compound of claim 1, wherein the compoundis selected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 11. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of the compound of claim 1, or apharmaceutically acceptable salt or stereoisomer thereof.
 12. A compoundhaving the formula I:

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:R₁ is: (a) NHCH₂CH₃, wherein the CH₂CH₃ is substituted with one NR₄R₅substituent; (b) NHCH₂phenyl, wherein the phenyl of CH₂phenyl issubstituted with one CH₃ substituent; (c) NH(phenyl), wherein the phenylis substituted with one, two, or three substituents independentlyselected from the group consisting of halogen, C₁-C₆ alkyl, C(halo)₃,C(O)NR₄R₅, NR₄R₅, OH, and OC₁-C₆ alkyl; or (d) NH(2-methylpyridin-4-yl);R₂ is C(O)OH; R₃ is H; each R₄ is an independently selected linear C₁-C₆alkyl; and each R₅ is an independently selected linear C₁-C₆ alkyl; orany R₄ and R₅, taken together with the nitrogen atom to which they areattached, independently forms a nitrogen-containing heterocyclyl,wherein each nitrogen-containing heterocyclyl is optionally andindependently substituted with one, two, or three substituentsindependently selected from the group consisting of halogen, C₁-C₆alkyl, C₁-C₆ trihaloalkyl, OH, and OC₁-C₆ alkyl.
 13. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of the compound of claim 12, or apharmaceutically acceptable salt or stereoisomer thereof.
 14. A compoundselected from the group consisting of:

or a pharmaceutically acceptable salt thereof.
 15. A pharmaceuticalcomposition comprising a pharmaceutically acceptable carrier and atherapeutically effective amount of the compound of claim 14, or apharmaceutically acceptable salt or stereoisomer thereof.